Extraction of high molecular weight DNA from microbial mats

Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functi...
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Autor*in:	Benjamin S. Bey [verfasserIn] Erin B. Fichot [verfasserIn] Gargi Dayama [verfasserIn] Alan W. Decho [verfasserIn] R. Sean Norman [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2010

Schlagwörter:	DNA extraction EPS hypersaline metagenomics microbial mats

Übergeordnetes Werk:	In: BioTechniques - Future Science Ltd, 2019, 49(2010), 3, Seite 631-640
Übergeordnetes Werk:	volume:49 ; year:2010 ; number:3 ; pages:631-640

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc Journal toc

DOI / URN:	10.2144/000113486

Katalog-ID:	DOAJ016945298

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520			\|a Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields <2 µg HMW DNA (<48 kb) per gram of mat sample, with A260:280 ratios <1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness.
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allfieldsGer	10.2144/000113486 doi (DE-627)DOAJ016945298 (DE-599)DOAJa600a63893184fe087c0e994a88ad2ae DE-627 ger DE-627 rakwb eng QH301-705.5 Benjamin S. Bey verfasserin aut Extraction of high molecular weight DNA from microbial mats 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields <2 µg HMW DNA (<48 kb) per gram of mat sample, with A260:280 ratios <1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness. DNA extraction EPS hypersaline metagenomics microbial mats Biology (General) Erin B. Fichot verfasserin aut Gargi Dayama verfasserin aut Alan W. Decho verfasserin aut R. Sean Norman verfasserin aut In BioTechniques Future Science Ltd, 2019 49(2010), 3, Seite 631-640 (DE-627)306320746 (DE-600)1496354-1 19409818 nnns volume:49 year:2010 number:3 pages:631-640 https://doi.org/10.2144/000113486 kostenfrei https://doaj.org/article/a600a63893184fe087c0e994a88ad2ae kostenfrei https://www.future-science.com/doi/10.2144/000113486 kostenfrei https://doaj.org/toc/0736-6205 Journal toc kostenfrei https://doaj.org/toc/1940-9818 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 49 2010 3 631-640
allfieldsSound	10.2144/000113486 doi (DE-627)DOAJ016945298 (DE-599)DOAJa600a63893184fe087c0e994a88ad2ae DE-627 ger DE-627 rakwb eng QH301-705.5 Benjamin S. Bey verfasserin aut Extraction of high molecular weight DNA from microbial mats 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields <2 µg HMW DNA (<48 kb) per gram of mat sample, with A260:280 ratios <1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness. DNA extraction EPS hypersaline metagenomics microbial mats Biology (General) Erin B. Fichot verfasserin aut Gargi Dayama verfasserin aut Alan W. Decho verfasserin aut R. Sean Norman verfasserin aut In BioTechniques Future Science Ltd, 2019 49(2010), 3, Seite 631-640 (DE-627)306320746 (DE-600)1496354-1 19409818 nnns volume:49 year:2010 number:3 pages:631-640 https://doi.org/10.2144/000113486 kostenfrei https://doaj.org/article/a600a63893184fe087c0e994a88ad2ae kostenfrei https://www.future-science.com/doi/10.2144/000113486 kostenfrei https://doaj.org/toc/0736-6205 Journal toc kostenfrei https://doaj.org/toc/1940-9818 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 49 2010 3 631-640
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source	In BioTechniques 49(2010), 3, Seite 631-640 volume:49 year:2010 number:3 pages:631-640
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Bey</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Extraction of high molecular weight DNA from microbial mats</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2010</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. 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callnumber-first	Q - Science
author	Benjamin S. Bey
spellingShingle	Benjamin S. Bey misc QH301-705.5 misc DNA extraction misc EPS misc hypersaline misc metagenomics misc microbial mats misc Biology (General) Extraction of high molecular weight DNA from microbial mats
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topic_title	QH301-705.5 Extraction of high molecular weight DNA from microbial mats DNA extraction EPS hypersaline metagenomics microbial mats
topic	misc QH301-705.5 misc DNA extraction misc EPS misc hypersaline misc metagenomics misc microbial mats misc Biology (General)
topic_unstemmed	misc QH301-705.5 misc DNA extraction misc EPS misc hypersaline misc metagenomics misc microbial mats misc Biology (General)
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title	Extraction of high molecular weight DNA from microbial mats
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title_full	Extraction of high molecular weight DNA from microbial mats
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title_sort	extraction of high molecular weight dna from microbial mats
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title_auth	Extraction of high molecular weight DNA from microbial mats
abstract	Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields <2 µg HMW DNA (<48 kb) per gram of mat sample, with A260:280 ratios <1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness.
abstractGer	Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields <2 µg HMW DNA (<48 kb) per gram of mat sample, with A260:280 ratios <1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness.
abstract_unstemmed	Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields <2 µg HMW DNA (<48 kb) per gram of mat sample, with A260:280 ratios <1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness.
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title_short	Extraction of high molecular weight DNA from microbial mats
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Extraction of high molecular weight DNA from microbial mats

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